Inhibition of azoreductase by oxygen. The role of the azo anion free radical metabolite in the reduction of oxygen to superoxide

Microsomal azo reduction of the diazonaphthol dye sulfonazo III is strongly inhibited by oxygen. The sulfonazo III anion radical metabolite, which is apparently the first intermediate of azoreductase activity, appears to react with oxygen in aerobic microsomal incubations. Even though the sulfonazo III is unchanged in aerobic NADPH-supplemented microsomal incubations, oxygen consumption increases 10-fold to 122 nmoles/min/mg of protein. This stimulated oxygen consumption is partially reversed by either superoxide dismutase or catalase. Concomitant with the increase in oxygen uptake is a 9-fold increase in the rate of superoxide formation. The oxidation of NADPH is also greatly increased by sulfonazo III, but is not influenced by superoxide dismutase or catalase. These results suggest that futile formation of the sulfonazo III anion free radical mediates oxygen reduction to O₂^- and NADPH oxidation, and that NADPH is not being oxidized by a superoxide-NADPH chain reaction.